Antisense inhibition of angiogenin expression

ABSTRACT

Disclosed are oligonucleotide compounds that inhibit the expression of angiogenin when administered to a mammal. Also disclosed are methods and pharmaceutical compositions for inhibiting the expression of angiogenin useful in therapy or diagnosis.

STATEMENT OF RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional ApplicationSerial No. 60/041,182 filed Mar. 21, 1997 hereby incorporated byreference in its entirety.

[0002] This application was funded in part by National Institutes ofHealth/National Cancer Institute grant no. ROI CA60046.

BACKGROUND OF THE INVENTION

[0003] 1. Field of the Invention

[0004] Embodiments of the present invention relate in general tocompositions and methods for inhibiting the expression of the angiogeningene thereby reducing the effects of angiogenin. Embodiments of thepresent invention also relate to inhibition of angiogenin geneexpression by antisense technologies including, but not limited to, theuse of antisense oligodeoxynucleotides and their derivatives.Embodiments of the present invention are further directed tocompositions and methods for detecting the angiogenin gene, as well asthe detection and diagnosis of abnormal expression of the angiogeningene in cells and tissues. Embodiments of the present invention are alsodirected to methods for inhibiting metastasis of cells, such as humantumor cells. Furthermore, this invention is directed to treatment ofconditions associated with abnormal angiogenesis, including cancer.

[0005] 2. Description of Related Art

[0006] Angiogenin is a potent inducer of angiogenesis [Fett, J. W.,Strydom, D. J., Lobb, R R, Aldermnn, E. M., Bethune, J. L., Riordan, J.F., and Vallee, B. L. (1985) Biochemistry 24, 5480-5486], a complexprocess of blood vessel formation that consists of several separate butinterconnected steps at the cellular and biochemical level: (i)activation of endothelial cells by the action of an angiogenic stimulus,(ii) adhesion and invasion of activated endothelial cells into thesurrounding tissues and migration toward the source of the angiogenicstimulus, and (iii) proliferation and differentiation of endothelialcells to form a new microvasculature [Folkman, J., and Shing, Y. (1992)J. Biol. Chem. 267, 10931-10934; Moscatelli, D., and Rifkin, D. B.(1988) Biochim. Biophys. Acta 948, 67-85]. Angiogenin has beendemonstrated to induce most of the individual events in the process ofangiogenesis including binding to endothelial cells [Badet, J., Soncin,F. Guitton, J. D., Lamare, O., Cartwright, T., and Barritault, D. (1989)Proc. Natl. Acad Sci. U.S.A. 86, 8427-8431], stimulating secondmessengers [Bicknell, R., and Vallee, B. L. (1988) Proc. Natl. Acad.Sci. U.S.A. 85, 5961-5965], mediating cell adhesion [Soncin, F. (1992)Proc. Natl. Acad. Sci. U.S.A. 89, 2232-2236], activating cell-associatedproteases [Hu, G-F., and Riordan, J. F. (1993) Biochem. Biophys. Res.Commun. 197, 682-687], inducing cell invasion [Hu, G -F., Riordan, J.F., and Vallee, B. L. (1994) Proc. Natl. Acad Sci. U.S.A. 91,12096-12100], inducing proliferation of endothelial cells [Hu, G -F.,Riordan, J. F., and Vallee, B. L. (1997) Proc. Natl. Acad. Sci. U.S.A.94, 2204-2209] and organizing the formation of tubular structures fromthe cultured endothelial cells [Jimi, S -I., Ito, K -I, Kohno, K., Ono,M., Kuwano, M., Itagaki, Y., and Isikawa, H. (1995) Biochem. Biophys.Res. Commun. 211, 476-483]. Angiogenin has also been shown to undergonuclear translocation in endothelial cells via receptor-mediatedendocytosis [Moroianu, J., and Riordan, J. F. (1994) Proc. Natl. Acad.Sci. U.S.A. 91, 1677-1681] and nuclear localization sequence-assistednuclear import [Moroianu, J., and Riordan, J. F. (1994) Biochem.Biophys. Res. Commun. 203, 1765-1772].

[0007] Although originally isolated from medium conditioned by humancolon cancer cells (Fett et al., 1985, supra) and subsequently shown tobe produced by several other histologic types of human tumors [Rybak, S.M., Fett, J. W., Yao, Q -Z., and Vallee, B. L. (1987) Biochem. Biophys.Res. Commun. 146, 1240-1248; Olson, K. A., Fett, J. W., French, T. C.,Key, M. E., and Vallee, B. L. (1995) Proc. Natl. Acad. Sci. U.S.A. 92,442-446], angiogenin also is a constituent of human plasma and normallycirculates at a concentration of 250 to 360 ng/ml [Shimoyama, S.,Gansauge, F., Gansauge, S., Negri, G., Oohara, T., and Beger, H. G.(1996) Cancer Res. 56, 2703-2706; Bläser, J., Triebl, S., Kopp, C., andTschesche, H. (1993) Eur. J. Clin. Chem. Clin. Biochem. 31, 513-516].

[0008] While angiogenesis is a tightly controlled process under usualphysiological conditions, abnormal angiogenesis can have devastatingconsequences as in pathological conditions such as arthritis, diabeticretinopathy and tumor growth. It is now well-established that the growthof virtually all solid tumors is angiogenesis dependent [Folkman, J.(1989) J. Natl. Cancer Inst. 82, 4-6]. Angiogenesis is also aprerequisite for the development of metastasis since it provides themeans whereby tumor cells disseminate from the original primary tumorand establish at distant sites [Mahadevan, V., and Hart, I. R. (1990)Rev. Oncol. 3, 97-103; Blood C. H., and Zetter B. R (1990) Biochim.Biophys. Acta 1032, 89-118]. Therefore, interference with the process oftumor-induced angiogenesis should be an effective therapy for bothprimary and metastatic cancers.

[0009] Several inhibitors of the functions of angiogenin have beendeveloped. These include: (i) monoclonal antibodies (mAbs) [Fett, J. W.,Olson, K. A., and Rybak, S. M. (1994) Biochemistry 33, 5421-5427], (ii)an angiogenin-binding protein [Hu, G -F, Chang, S -L Riordan J. F., andVallee, B. L. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 2227-2231; Hu, G-F., Strydom, D. J., Fett, J. W., Riordan, J. F., and Vallee B. L.(1993) Proc. Natl. Acad. Sci. U.S.A. 90, 1217-1221; Moroianu, J., Fett,J. W., Riordan, J. F., and Vallee B. L. (1993) Proc. Natl. Acad Sci.U.S.A. 90, 3815-3819], (iii) the placental ribonuclease inhibitor (PRI)[Shapiro, R., and Vallee, B. L. (1987) Proc. Natl. Acad Sci. U.S.A.84,2238-2241], (iv) peptides synthesized based on the C-terminalsequence of angiogenin [Rybak, S. M., Auld, D. S., St. Clair, D. K.,Yao, Q -Z., and Fett, J. W. (1989) Biochem. Biophys. Res. Commun. 162,535-543], and (v) inhibitory site-directed mutants of angiogenin[Shapiro, R., and Vallee, B. L. (1989) Biochemistry 28, 7401-7408]. Allinhibit angiogenin's activities but are not directly cytotoxic to humantumor cells grown in tissue culture.

[0010] mAbs or the angiogenin-binding protein when administered locallyinto xenografts of human tumor cells that were injected subcutaneously(s.c.) into athymic mice are able to delay or, remarkedly, completelyprevent the appearance of colon, lung and fibrosarcoma tumors in theseanimals [Olson et al., 1995, supra, Olson, K. A, French, T. C., Vallee,B. L., and Fett, J. W. (1994) Cancer Res. 54, 4576-4579]. Histologicalexamination revealed that the mechanism of tumor growth inhibition wasvia an anti-angiogenesis mechanism (Olson et al., 1995, supra). Thus,the inactivation of tumor-produced angiogenin or inhibition ofexpression of the angiogenin gene by tumor cells promise to be apowerful means of managing cancer, either alone or in combination withmore conventional therapies (i.e., chemotherapy, radiotherapy,immunotherapy, etc.).

[0011] Expression of specific genes may be suppressed byoligonucleotides having a nucleotide sequence complementary to the mRNAtranscript of the target gene thereby selectively impeding translationand has been termed an “antisense” methodology. In addition, “antigene”or “triplex” methodologies may also suppress expression of genes byusing an oligonucleotide which is complementary to a selected site ofdouble stranded DNA thereby forming a triple-stranded complex toselectively inhibit transcription of the gene. Both “antisense” and“antigene” methodologies find utility as molecular tools for geneticanalysis. Antisense oligonucleotides have been extensively used toinhibit gene expression in normal and abnormal cells in studies of thefunction of various cell proteins. Major advances have been made in thedevelopment of antisense or antigene reagents for the treatment ofdisease states in animals and humans [“Antisense Therapeutics” Agrawal,S. (ed.), Humana Press, 1996; Crooke, S. T., and Bennett, C. F. (1996)Annu. Rev. Pharmacol. Toxicol. 36, 107-129; “Prospects for theTherapeutic Use of Antigene Oligonucleotides”, Maher, L.J. (1996) CancerInvestigation 14(1), 66-82 each hereby incorporated by reference in itsentirety].

[0012] As therapeutics, oligonucleotides possess two major requirementsfor successful drug design—specificity and affinity. These are achievedby selectively targeting particular DNA or RNA sequences exploitingWatson-Crick base pairing with resulting interference of proteinproduction whether through inhibition of gene transcription ortranslation of mRNA. This approach allows for rapid identification oflead compounds based on knowledge of a relevant gene target species.Recently, improvements have been made in increasing both the stabilityand affinity of these compounds. Phosphorothioate analogs ofoligodeoxynucleotides (ODNs), in which nonbridging phosphoryl oxygens inthe backbone of DNA are substituted with sulfur, abbreviated [S]ODNs,are substantially more stable than their native phosphodiestercounterparts, while other derivatives, such as those alkylated on sugaroxygen groups, show enhanced target affinity. [S]ODNs possess goodbiological activity, pharmacology, pharmacokinetics and safety in vivo(Agrawal, 1996, supra, and references therein) and have been usedsuccessfully for anti-tumor therapy in animal models (Crooke andBennett, 1996, supra). Antisense reagents are now in clinical trials fortreatment of cancers and viral infections (Agrawal, 1996, supra).Successful inhibition of specific gene function has been achieved bytargeting various sites on specific mRNA sequences that include the AUGtranslational initiation codon, 5′-transcriptional start site,3′-termination codon and sequences in both the 5′- and 3′-untranslatedregions. Experience to date has indicated that success has been achievedby targeting these and other regions.

[0013] As examples, U.S. Pat. No. 5,098,890 is directed to antisenseoligonucleotides complementary to the c-myb oncogene and antisenseoligonucleotide therapies for certain cancerous conditions. U.S. Pat.No. 5,135,917 provides antisense oligonucleotides that inhibit humaninterleukin-1 receptor expression. U.S. Pat. No. 5,087,617 providesmethods for treating cancer patients with antisense oligonucleotides.U.S. Pat. No. 5,166,195 provides oligonucleotide inhibitors of HIV. U.S.Pat. No. 5,004,810 provides oligomers capable of hybridizing to herpessimplex virus Vmw65 mRNA and inhibiting replication. U.S. Pat. No.5,194,428 provides antisense oligonucleotides having antiviral activityagainst influenzavirus. U.S. Pat. No. 4,806,463 provides antisenseoligonucleotides and methods using them to inhibit HTLV-III replication.U.S. Pat. No. 5,286,717 is directed to a mixed linkage oligonucleotidephosphorothioates complementary to an oncogene. U.S. Pat. No. 5,276,019and U.S. Pat. No. 5,264,423 are directed to phosphorothioateoligonucleotide analogs used to prevent replication of foreign nucleicacids in cells.

[0014] The nucleic acid sequence of the entire angiogenin gene includingthe 5′- and 3′-flanking regions has been determined [Kurachi, K., Davie,E. W., Strydom, D. J. Riordan, J. F. and Vallee, B. L. (1985)Biochemistry 24, 5494-5499 hereby incorporated by reference in itsentirety]. The native DNA segment coding for angiogenin, as all suchmammalian DNA strands, has two strands; a sense strand and an antisensestrand held together by hydrogen bonding. The messenger RNA coding forangiogenin has the same nucleotide sequence as the sense strand exceptthat the DNA thymidine is replaced by uridine. Thus, synthetic antisensenucleotide sequences should bind with the DNA and RNA coding forangiogenin.

[0015] However, it is unknown whether antisense reagents will in fact beeffective for inhibition of angiogenin expression. To date, nooligonucleotide antisense reagents have been designed or demonstrated tobe useful in the inhibition of the expression of angiogenin.Accordingly, a need exists to discover oligonucleotide antisensereagents which can prove useful in modulating or inhibiting theexpression of angiogenin and to further discover methods by which sucholigonucleotide antisense reagents can be used in methods of diagnosisand treatment.

SUMMARY OF THE INVENTION

[0016] Embodiments of the present invention are based on the discoveryof oligonucleotide reagents capable of targeting nucleic acid sequencesencoding angiogenin in a manner to inhibit (i.e., reduce, eliminate orotherwise interfere with) the expression of angiogenin. Eacholigonucleotide, or analog thereof, has a nucleotide or base sequencewhich is complementary, i.e. capable of hybridizing with or binding to,at least a target portion of the nucleic acid encoding angiogenin, i.e.the angiogenin gene DNA or RNA, which has significance in expressingangiogenin. In accordance with one aspect of the present invention,targeted RNA or DNA, or cells containing it are contacted witholigonucleotide or analogs thereof which are configured to bind to theRNA or DNA in a manner to inhibit the expression of angiogenin whetherby interfering with gene transcription as in an antigene strategy or byinterfering with translation of mRNA as in an antisense strategy.

[0017] Embodiments of the present invention are further directed tomethods for inhibiting the expression of angiogenin in a mammal byadministering to or otherwise treating the mammal with an effectiveamount of an oligonucleotide or analog thereof having a base sequencecomplementary to a target portion of a nucleic acid encoding angiogeninso as to inhibit the expression of angiogenin. Embodiments of thepresent invention are also directed to methods for reducing size oftumors associated with angiogenesis in a mammal comprising administeringto the mammal an effective amount of an oligonucleotide or analogthereof having a base sequence complementary to a target portion of anucleic acid encoding angiogenin so as to reduce tumor size. Embodimentsof the present invention are further directed to methods for decreasingproduction of angiogenin in a mammal comprising administering to themammal an effective amount of an oligonucleotide or analog thereofhaving a base sequence complementary to a target portion of a nucleicacid encoding angiogenin so as to decrease production of angiogenin.Embodiments of the present invention are still further directed tomethods for inhibiting metastasis of tumor cells in a mammal comprisingadministering to the mammal an effective amount of an oligonucleotide oranalog thereof having a base sequence complementary to a target portionof a nucleic acid encoding angiogenin so as to inhibit metastasis oftumor cells. Embodiments of the present invention are even still furtherdirected to methods for inhibiting the establishment of tumor cells in amammal comprising administering to the mammal an effective amount of anoligonucleotide or analog thereof having a base sequence complementaryto a target portion of a nucleic acid encoding angiogenin so as toinhibit establishment of tumor cells. Embodiments of the presentinvention are even still further directed to methods for inhibitinggrowth of tumors associated with angiogenesis in a mammal comprisingadministering to the mammal an effective amount of an oligonucleotide oranalog thereof having a base sequence complementary to a target portionof a nucleic acid encoding angiogenin so as to inhibit tumor growth. Theoligonucleotides, analogs thereof and methods described herein aretherefore useful in methods of therapeutically treating a mammal,including a human, afflicted with pathological conditions associatedwith abnormal or unwanted angiogenesis, including cancer.

[0018] As an alternate embodiment of the present invention, labeledoligonucleotides may also be useful for diagnosing conditions associatedwith abnormal angiogenin expression since the labeled oligonucleotidesof the present invention can also bind to the angiogenin gene, DNA orRNA and then can be detected and/or measured. Alternate embodiments ofthe present invention include methods detecting the presence ofangiogenin in a sample comprising contacting the sample with a labeledoligonucleotide or analog thereof having a base sequence complementaryto a target portion of a nucleic acid encoding angiogenin, allowing thelabeled oligonucleotide or analog thereof to bind to the target portionof the nucleic acid encoding angiogenin, and detecting the labeledoligonucleotide or analog thereof. A further alternate embodiment of thepresent invention includes methods for detecting the presence ofangiogenin in a mammal comprising administering to the mammal a labeledoligonucleotide or analog thereof having a base sequence complementaryto a target portion of a nucleic acid encoding angiogenin, allowing thelabeled oligonucleotide or analog thereof to bind to the target portionof the nucleic acid encoding angiogenin, and detecting the labeledoligonucleotide or analog thereof A still further alternate embodimentof the present invention includes methods for diagnosing conditionsassociated with abnormal angiogenesis in a mammal comprisingadministering to the mammal a labeled oligonucleotide or analog thereofhaving a base sequence complementary to a target portion of a nucleicacid encoding angiogenin, allowing the labeled oligonucleotide or analogthereof to bind to the target portion of the nucleic acid encodingangiogenin, detecting the labeled oligonucleotide or analog thereofmeasuring the labeled oligonucleotide or analog thereof, and determiningthe abnormal condition based on the detecting and measuring of thelabeled oligonucleotide or analog thereof

[0019] These and other objects, features and advantages of the presentinvention will become apparent by reference to the remaining portions ofthe specification and the attached drawings.

BRIEF DESCRIPTION OF DRAWINGS

[0020] In the course of the detailed description of certain preferredembodiments to follow, reference will be made to the attached drawings,in which,

[0021]FIG. 1 depicts the nucleic acid sequence of the entire humanangiogenin gene including the cDNA sequence as identified by arrows.

[0022]FIG. 2 is a graph depicting the inhibition by angiogenin antisense[S]ODN JF2S of angiogenin expression by PC-3 tumor cells in vitro andtheir subsequent growth in vivo.

[0023]FIG. 3 is a graph depicting the inhibition by angiogenin antisense[S]ODN JF2S of angiogenin expression by HT-29 tumor cells in vitro andtheir subsequent growth in vivo.

[0024]FIG. 4 is a graph showing treatment of HT-29 tumor cells in vitrowith antisense [S]ODN JF2S and control sense [S]ODN JF1S and theirsubsequent growth in vivo.

[0025]FIG. 5 is a graph showing treatment of PC-3 tumor cells in vitrowith antisense [S]ODN JF2S and control sense [S]ODN JF1S and theirsubsequent growth in vivo.

[0026]FIG. 6 is a graph showing treatment of MDA-MB435 tumor cells invitro with antisense [S]ODN JF2S and control sense [S] ODN JF1S andtheir subsequent growth in vivo.

[0027]FIG. 7 is a graph showing treatment of PC-3M tumor cells in vitrowith antisense [S]ODN JF2S and control sense [S]ODN JF1S and theirsubsequent growth in vivo.

[0028]FIG. 8 is a photograph showing the differences in the presence andsize of angiogenin antisense [S]ODN (JF2S) and control(lipofectin)-treated PC-3 tumors excised from athymic mice.

[0029]FIG. 9 is a photograph showing the differences in the presence andsize of angiogenin antisense [S]ODN (JF2S) and control(lipofectin)-treated HT-29 tumors excised from athymic mice.

[0030]FIG. 10 is a photograph showing the differences in the presenceand size of angiogenin antisense [S]ODN (JF2S), control sense [S]ODN(JF1S) and control (lipofectin)-treated HT-29 tumors excised fromathymic mice.

[0031]FIG. 11 is a photograph showing the differences in the presenceand size of angiogenin antisense [S]ODN (JF2S), control sense [S]ODN(JF1S) and control (lipofectin)-treated PC-3 tumors excised from athymicmice.

[0032]FIG. 12 is photograph showing the differences in the presence andsize of angiogenin antisense [S]ODN (JF2S), control sense [S]ODN (JF1S)and control (lipofectin)-treated MDA-MB-435 tumors excised from athymicmice.

[0033]FIG. 13 is a photograph showing the differences in the presenceand size of angiogenin antisense [S]ODN (JF2S), control sense [S]ODN(JF1S) and control (lipofectin)-treated PC-3M tumors excised fromathymic mice.

[0034]FIG. 14 is a graph showing inhibition of the expression ofangiogenin by PC-3 and PC-3M tumor cell lines in culture using the twoangiogenin antisense [S]ODNs, JF2S and JF4S.

[0035]FIG. 15 is a graph showing in vivo therapy of PC-3 tumors withangiogenin antisense [S]ODN JF2S, control sense [S]ODN JF1S and PBSdiluent control in three separate experiments.

[0036]FIG. 16 is a graph showing in vivo therapy of MDA-MB-435 tumorswith angiogenin antisense [S]ODN JF2S, control sense [S]ODN JF1S and PBSdiluent control.

[0037]FIG. 17 is a graph showing in vivo therapy of MCF-7 tumors withangiogenin antisense [S]ODN JF2S, control sense [S]ODN JF1S and PBSdiluent control.

DETAILED DESCRIPTION OF CERTAIN PREFERRED EMBODIMENTS

[0038] The principles of the present invention may be advantageouslyapplied to produce novel oligonucleotides or analogs thereof which bindto or otherwise target nucleic acids encoding angiogenin. Theoligonucleotides or analogs thereof interfere with the normal functionof the nucleic acids and otherwise inhibit the transcription,replication or translation associated with the expression of angiogenin.

[0039] Angiogenesis is prominent in solid tumor formation andmetastasis. Angiogenic factors have been found associated with severalsolid tumors such as rhabdomyosarcomas, retinoblastoma, Ewing sarcoma,neuroblastoma, and osteosarcoma. A tumor cannot expand without a bloodsupply to provide nutrients and remove cellular wastes. Tumors in whichangiogenesis is important include solid tumors, and benign tumors suchas acoustic neuroma, neurofibroma, trachoma and pyogenic granulomas. Thepresent invention is directed towards prevention of angiogenesis in thetreatment of these and other angiogenesis dependent tumors and theresultant damage to the mammal due to the presence of the tumor.

[0040] Angiogenesis is also associated with blood-born tumors such asleukemias, any of various acute or chronic neoplastic diseases of thebone marrow in which unrestrained proliferation of white blood cellsoccurs, usually accompanied by anemia, impaired blood clotting, andenlargement of the lymph nodes, liver, and spleen. It is believed thatangiogenesis plays a role in the abnormalities in the bone marrow thatgives rise to leukemia-like tumors.

[0041] Angiogenesis is important in two stages of tumor metastasis. Thefirst stage where angiogenesis stimulation is important is in thevascularization of the tumor which allows cells to enter the bloodstream and to circulate throughout the body. After the tumor cells haveleft the primary site, and have settled into the secondary, metastasissite, angiogenesis must occur before the new tumor can grow and expand.Therefore, embodiments of the present invention are directed to theinhibition of angiogenesis as a treatment for the prevention ofmetastasis of tumors and containment of the neoplastic growth at theprimary site.

[0042] Examples of diseases mediated by angiogenesis are disclosed inthe prior art such as U.S. Pat. No. 5,712,291 and include ocularneovascular disease as well as the other diseases to follow. Ocularneovascular disease is characterized by invasion of new blood vesselsinto the structure of the eye such as the retina or cornea. It is themost common cause of blindness and is involved in approximately twentyeye diseases. In age-related macular degeneration, the associated visualproblems are caused by an ingrowth of choroidal capillaries throughdefects in Bruch's membrane with proliferation of fibrovascular tissuebeneath the retinal pigment epithelium. Angiogenic damage is alsoassociated with diabetic retinopathy, retinopathy of prematurity, comealgraft rejection, neovascular glaucoma and retrolental fibroplasia. Otherdiseases associated with comeal neovascularization include, but are notlimited to, epidemic keratoconjunctivitis, Vitamin A deficiency, contactlens overwear, atopic keratitis, superior limbic keratitis, pterygiumkeratitis sicca, sjogrens, acne rosacea, phylectenulosis, syphilis,mycobacteria infections, lipid degeneration, chemical burns, bacterialulcers, fungal ulcers, Herpes simples infections, Herpes zosterinfections, protozoan infections, Kaposi sarcoma, Mooren ulcer,Terrien's marginal degeneration, marginal keratolysis, rheumatoidarthritis, systemic lupus, polyarteritis, trauma, Wegener's sarcoidosis,Scleritis, Steven Johnson's disease, periphigoid radical keratotomy, andcorneal graph rejection.

[0043] Diseases associated with retinal/choroidal neovascularizationinclude, but are not limited to, diabetic retinopathy, maculardegeneration, sickle cell anemia, sarcoid, syphilis, pseudoxanthomaelasticum, Pagets disease, vein occlusion, artery occlusion, carotidobstructive disease, chronic uveitis/vitritis, mycobacterial infections,Lyme's disease, systemic lupus erythematosis, retinopathy ofprematurity, Eales disease, Bechets disease, infections causing aretinitis or choroiditis, presumed ocular histoplasmosis, Bests disease,myopia, optic pits, Stargarts disease, pars planitis, chronic retinaldetachment, hyperviscosity syndromes, toxoplasmosis, trauma andpost-laser complications. Other diseases include, but are not limitedto, diseases associated with rubeosis (neovascularization of the angle)and diseases caused by the abnormal proliferation of fibrovascular orfibrous tissue including all forms of proliferative vitreoretinopathy.

[0044] Another disease in which angiogenesis is believed to be involvedis rheumatoid arthritis. The blood vessels in the synovial lining of thejoints undergo angiogenesis. In addition to forming new vascularnetworks, the endothelial cells release factors and reactive oxygenspecies that lead to pannus growth and cartilage destruction. Thefactors involved in angiogenesis may actively contribute to, and helpmaintain, the chronically inflamed state of rheumatoid arthritis.

[0045] Factors associated with angiogenesis may also have a role inosteoarthritis. The activation of the chondrocytes by angiogenic-relatedfactors contributes to the destruction of the joint. At a later stage,the angiogenic factors would promote new bone formation. Therapeuticintervention that prevents the bone destruction could halt the progressof the disease and provide relief for persons suffering from arthritis.

[0046] Chronic inflammation may also involve pathological angiogenesis.Such disease states as ulcerative colitis and Crohn's disease showhistological changes with the ingrowth of new blood vessels into theinflamed tissues. Bartonellosis, a bacterial infection found in SouthAmerica, can result in a chronic stage that is characterized byproliferation of vascular endothelial cells. Another pathological roleassociated with angiogenesis is found in atherosclerosis. The plaquesformed with the lumen of blood vessels have been shown to haveangiogenic stimulatory activity.

[0047] One of the most frequent angiogenic diseases of childhood is thehemangioma. In most cases, the tumors are benign and regress withoutintervention. In more severe cases, the tumors progress to largecavernous and infiltrative forms and create clinical complications.Systemic forms of hemangiomas, the hemangiomatoses, have a highmortality rate. Therapy-resistant hemangiomas exist that cannot betreated with therapeutics currently in use.

[0048] Angiogenesis is also responsible for damage found in hereditarydiseases such as Osler-Weber-Rendu disease, or hereditary hemorrhagictelangiectasia. This is an inherited disease characterized by multiplesmall angiomas, tumor of blood or lymph vessels. The angiomas are foundin the skin and mucous membranes, often accompanied by epistaxis(nosebleeds) or gastrointestinal bleeding sometimes with pulmonary orhepatic arteriovenous fistula.

[0049] Embodiments of the present invention further include treatment ofthe above disease states through the inhibition of angiogenesis

[0050] The relationship between an oligonucleotide and its complementarynucleic acid target to which it hybridizes is commonly referred to as“antisense” if the complementary nucleic acid target is single strandedor “antigene” or “triplex” if the complementary nucleic acid target isdouble stranded. It is to be understood that the oligonucleotides andmethods of the present invention described herein are useful in bothantisense or antigene approaches. Accordingly, those terms are usedinterchangeably herein.

[0051] In accordance with the teachings of the present invention, theoligonucleotide employed in the methods of the present invention willgenerally have a sequence that is complementary to the sequence of thetarget nucleic acid whether that be in the form of single stranded RNAor DNA or double stranded DNA. “Targeting” an oligonucleotide to anucleic acid sequence of the angiogenin gene includes determining a siteor sites within the nucleic acid sequence for the oligonucleotideinteraction to occur such that the inhibition of the expression ofangiogenin will result. “Inhibition of the expression of angiogenin” isherein defined as that phrase is normally understood and to also includethe elimination of, prevention of, reduction of or other interferencewith the expression of angiogenin occurring prior to or in the absenceof the interaction between the oligonucleotide and the nucleic acidsequence of the angiogenin gene. “Inhibition” itself is herein definedas the elimination of, prevention of; reduction of or other interferencewith the particular mechanism being interfered with. Once the desiredtarget site or sites have been identified anywhere along the entirenucleic acid sequence of the angiogenin gene, one or moreoligonucleotides are chosen which are sufficiently complementary to thetarget, i.e. hybridize sufficiently well and with sufficientspecificity, to inhibit the expression of angiogenin.

[0052] The terms “hybridization” or “to bind” as used herein meanshydrogen bonding, also known as Watson-Crick base pairing, betweencomplementary bases (i.e. purines or pyrimidines), usually on oppositenucleic acid strands or two regions of a nucleic acid strand. Guanineand cytosine are examples of complementary bases which are known to formthree hydrogen bonds between them. Adenine and thymine are examples ofcomplementary bases which form two hydrogen bonds between them.

[0053] The letters A, G, C, T, and U respectively indicate nucleotidesin which the nucleoside is adenosine, guanosine, cytidine, thymidine,and uridine. As used herein, oligonucleotides that are antisense to thetarget angiogenin nucleic acid sense strand are oligonucleotides whichhave a nucleoside sequence complementary to the sense strand. Table 1shows the four possible sense strand bases and their complements presentin an antisense compound. TABLE 1 Sense Antisense Adenine ThymineGuanine Cytosine Cytosine Guanine Thymine Adenine

[0054] “Specifically hybridizable” and “complementary” are terms whichare used to indicate a sufficient degree of complementarity such thatstable and specific binding occurs between the target nucleic acid andthe oligonucleotide. It is to be understood that an oligonucleotide neednot be 100% complementary to its target nucleic acid to be specificallyhybridizable, i.e. it may lack one or more complements for certainnucleotides in the targeted nucleic acid sequence. An oligonucleotide isspecifically hybridizable when binding of the oligonucleotide to thetarget nucleic acid inhibits the normal function of the target nucleicacid to cause a loss of utility whether of transcription or translation,and there is a sufficient degree of complementarity to avoidnon-specific binding of the oligonucleotide to non-target sequencesunder conditions in which specific binding is desired, i.e. underphysiological conditions in the case of in vivo assays or therapeutictreatment, or in the case of in vitro assays, under conditions in whichthe assays are conducted. Accordingly, absolute complementarity is notrequired in the practice of the present invention. In general, anyoligonucleotide having sufficient complementarity to form a stableduplex with the target single stranded RNA or DNA or a stable triplexwith the target double stranded DNA is considered to be suitable. Sincestable duplex or triplex formation depends on the sequence and length ofthe hybridizing oligonucleotide and the degree of complementaritybetween the antisense oligonucleotide and the target sequence, thesystem can tolerate less fidelity (complementarity) when longeroligonucleotides are used. In short, any interaction or binding of anoligonucleotide or oligonucleotide analog with a target nucleic acidencoding angiogenin is believed to have the potential to inhibit theexpression of angiogenin.

[0055] In the context of this invention, the term “oligonucleotide”refers to a plurality of joined nucleotide units formed fromnaturally-occurring bases and ribofuranosyl groups joined by nativephosphodiester bonds. This term effectively refers tonaturally-occurring species or synthetic species formed fromnaturally-occurring subunits and includes both oligomers ofribonucleotide i.e., oligoribonucleotides, and oligomers ofdeoxyribonucleotide i.e, oligodeoxyribonucleotides (also referred toherein as “oligodeoxynucleotides”). As used herein, unless otherwiseindicated, the term “oligonucleotide” also includes oligomers which maybe large enough to be termed “polynucleotides”. As further used herein,the terms “oligonucleotide” and “oligodeoxynucleotide” include not onlyoligomers and polymers of the biologically significant nucleotides, i.e.nucleotides of adenine (“A”), deoxyadenine (“dA”), guanine (“G”),deoxyguanine (“dG”), cytosine (“C”) deoxycytosine (“dC”), thymine (“T”)and uracil (“U”), but also oligomers and polymers hybridizable toangiogenin DNA or RNA which may contain other nucleotides.

[0056] “Oligonucleotide analog” as that term is used in connection withthis invention, refers to a compound having a modified internucleotidelinkage, a modified purine or pyrimidine moiety, a modified sugarmoiety, a modified 5′ hydroxyl moiety, a modified 3′ hydroxyl moiety ora modified 2′ hydroxyl moiety. The analogs including the modifiedmoieties function similarly to oligonucleotides in that they hybridizeor otherwise bind to target nucleic acids but which have nonnaturally-occurring portions wherein one or more purine or pyrimidinemoieties, sugar moieties or internucleotide phosphate linkages ischemically modified, for example, to improve stability and/or lipidsolubility to enhance the ability of the oligonucleotides to penetrateinto the region of cells where the RNA whose activity is to be modulatedis located. For example, it is known that enhanced lipid solubilityand/or resistance to nuclease digestion results by substituting a methylgroup or sulfur atom for a phosphate oxygen in the internucleotidephosphodiester linkage. Exemplary among these are the phosphorothioateand other sulfur containing species which are known in the art.Phosphorothioates are compounds in which one of the non-bridging oxygenatoms in the phosphate portion of the nucleotide is replaced by sulfur.These phosphorothioates are stable to cleavage by nucleases, and sincethey have the same number of charges as normal oligodeoxynucleotides,they have good aqueous solubility. Other modified oligonucleotides oranalogs such as alkyl phosphorothioate, phosphodiester, phosphotriester,N-alkyl phosphoramidates, phosphorodithioates, alkyl phosphonates, andshort chain alkyl or cycloalkyl structures may also be useful. Inaccordance with other preferred embodiments, one or more phosphodiesterbonds are substituted with structures which are, at once, substantiallynon-ionic and non-chiral to produce mixed linkage oligonucleotides.Persons of ordinary skill in the art will be able to select otherlinkages for use in the practice of the invention.

[0057] Oligonucleotide analogs may also comprise altered base or sugarunits, have charged or uncharged backbones, have additions at the endsof the oligonucleotide molecule or other modifications consistent withthe spirit of this invention. Such analogs are best described as beingfunctionally interchangeable with natural oligonucleotides (orsynthesized oligonucleotides along natural lines), but which have one ormore differences from natural structure. All such analogs arecomprehended by this invention so long as they can function effectivelyto bind to selected portions of nucleic acids encoding angiogenin.

[0058] In accordance with the principles of the present invention,oligonucleotides complementary to and hybridizable with any portion ofnucleic acids responsible for expression of angiogenin whether human oranimal are, in principle, effective for inhibiting the expression ofangiogenin in the respective mammal. It is therefore to be understoodthat the principles of the present invention apply to all mammals,including humans, where inhibition of the expression of angiogenin isdesired. For example, the nucleic acid sequence encoding mouseangiogenin is known. See Bond, M. D., and Vallee, B. L. (1990) Biochem.Biophys. Res. Commun. 171, 988-995. The nucleic acid sequence for humanangiogenin is shown in FIG. 1. Accordingly, the application of theprinciples of the present invention not only include human uses, butanimal uses as well.

[0059] Oligonucleotides according to certain embodiments of the presentinvention are represented by Formula I below although additionalembodiments are described throughout this disclosure:

[0060] in which

[0061] X is O, S, or C₁₋₄ alkyl;

[0062] B is adenine, guanine, cytosine, or thymine selected such thatthe oligonucleotide has a complementary base sequence with a portion ofthe nucleic acid strand coding for angiogenin thereby inhibitingexpression thereof;

[0063] R, is H, C₁₋₄ alkyl or substituted acridine;

[0064] R₂ is H, OH, SH, F, OCH₃, OCN, or OCU₆CH₃; and

[0065] n is 5to 100.

[0066] Oligonucleotides within the scope of the present invention,including those represented by Formula I, include pharmaceuticallyacceptable salts or hydrates thereof. Oligonucleotides within the scopeof the present invention optionally may include intercalating moleculesor ribozyme sequences and may optionally have intervening sequences ofother nucleotides or non-nucleotide molecules provided that sucholigonucleotides hybridize with angiogenin DNA or RNA and inhibit itsexpression.

[0067] While any length oligonucleotide may be utilized in the practiceof the invention, such as an oligonucleotide complementary to the entireangiogenin gene, oligonucleotides having between 5 to 100 subunits findutility and are preferred in the practice of the present invention. Itis preferred that such oligonucleotides and analogs comprise at leastabout 5 subunits with from about 8 to 50 subunits being more preferred.As will be appreciated, a subunit is a base and sugar combinationsuitably bound to adjacent subunits through phosphodiester or othermodified bonds as previously discussed.

[0068] Oligonucleotides shorter than 15 bases may be less specific inhybridizing to the target angiogenin mRNA, and may be more easilydestroyed by enzymatic digestion. Hence, oligonucleotides having 15 ormore nucleotides are preferred. Sequences longer than 18 to 25nucleotides may be somewhat less effective in inhibiting angiogenintranslation because of decreased uptake by the target cell. Thus,oligomers of 15-25 nucleotides are most preferred in the practice of thepresent invention, particularly oligomers of 15-18 nucleotides.

[0069] It is to be understood that oligonucleotides having a sequencecomplementary to any region of the angiogenin gene find utility in thepresent invention, however oligodeoxynucleotides complementary to aportion of (i) the “AUG” translational start site, (Hi) the5′-transcription initiation site, (iii) the 5′-“TATA” box site and, (iv)the 3′-termination site are particularly preferred. Random sequences inboth the 5′-untranslated and 3′-untranslated regions are also usefultarget nucleic acids for designing oligonucleotides for the inhibitionof the expression of angiogenin.

[0070] Oligonucleotides of the present invention, including thoserepresented by Formula I, hybridize or otherwise bind to target nucleicacids encoding for angiogenin, the entire gene sequence of which isshown in FIG. 1. When X in Formula 1 is oxygen, the nucleotides areconnected by phosphodiester bonds. However, oligonucleotides of thepresent invention include analogs which differ from native DNA in thatsome or all of the phosphates in the nucleotides are replaced byphosphorothioates (in the case of X being sulfur), methylphosphonates(in the case of X being CH₃) or other C₁₋₄ alkylphosphonates such asethyl, propyl, butyl, methyl phosphonate analogs disclosed by U.S. Pat.No. 4,469,863, phosphonate modified oligodeoxynucleotides described byLaPlanche, et al. Nucleic Acid Research 14:9081 (1986) and by Stec. etal. J. Am Chem. Soc. 106:6077 (1984), phosphodiesters andphosphotriesters. These compounds are referred to herein as having amodified oligonucleotide linkage moiety. Furthermore, recent advances inthe production of oligoribonucleotide analogues mean that other agentsmay also be used for the purposes described here, e.g.2′-methylribonucleotides (Inoue et al. Nucleic Acids Res. 15,6131, 1987)and chimeric oligonucleotides that are composite RNA-DNA analogues(Inoue et al. FEBS Lett. 215, 327, 1987).

[0071] Specific examples of some preferred oligonucleotides envisionedfor this invention may contain phosphorothioates, phosphotriesters,methyl phosphonates, short chain alkyl or cycloalkyl intersugar linkagesor short chain heteroatomic or heterocyclic intersugar (“backbone”)linkages. Most preferred are phosphorothioates and those withCH₂—NH—O—CH₂, CH₂—N(CH₃)—O—CH₂, CH₃—O—NlCH₃)—CH₂, CH₂—N(CH₃)—N(CH₃)—CH₂and O—N(CH₃)—CH₂—CH₂ backbones (where phosphodiester is O—P—O—CH₂). Alsopreferred are oligonucleotides having morpholino backbone structures.Summerton, J. E. and Weller, D. D., U.S. Pat. No. 5,034,506. In otherpreferred embodiments, such as the protein-nucleic acid orpeptide-nucleic acid (PNA) backbone, the phosphodiester backbone of theoligonucleotide may be replaced with a polyamide backbone, the basesbeing bound directly or indirectly to the aza nitrogen atoms of thepolyamide backbone. P. E. Nielsen, M. Egholm, R. H. Berg, O. Buchardt,Science 1991, 154, 1497.

[0072] The oligonucleotides of Formula I optionally may be furtherdifferentiated from native DNA by replacing one or both of the freehydroxy groups with C₁₋₄ alkoxy groups (in the case of R₁ being C₁₋₄alkyl). As used herein, C₁₋₄ alkyl means a branched or unbranchedhydrocarbon having 1 to 4 carbon atoms.

[0073] Formula I oligonucleotides may also be substituted at the 3′and/or 5′ ends by R₁ being an intercalating agent such as a “substitutedacridine” which means any acridine derivative capable of intercalatingnucleotide strands such as DNA. Preferred substituted acridines are2-methoxy-6-chloro-9-pentylaminoacridine,N-(6-chloro-2-methoxyacridinyl)-O-methoxydisopropylaminophosphinyl-3-aminopropanoland N-(6chloro-2-methoxyacridinyl)-O-methoxydisopropylaminophosphinyl-5-aminopentanol.Other suitable acridine derivatives are readily apparent to personsskilled in the art.

[0074] Formula I oligonucleotides may also include ribozyme sequencesinserted into their nucleotide sequence. The ribozyme sequences areinserted into Formula I compounds such that they are immediatelypreceded by AUC, UUC, GUA, GUU, GUC, or, preferably, CUC. The ribozymesequence is any sequence which can be inserted and causes self-cleavageof messenger RNA- The sequence CUG AUG AGU CCG UGA CGA A is preferred.Other such sequences can be prepared as described by Haseloff andGerlach, Nature (Aug. 18, 1988) 334; 585-591.

[0075] It is generally preferred for use in some embodiments of thisinvention that the 2′ position of the linking sugar moieties in at leastsome of the subunits of the oligonucleotides or oligonucleotide analogsbe substituted, Thus, 2′ substituents such as R₂ is OH, SH, SCH₂, OCH₃,F, OCN, OCH₆CH₃, OCH₃OCH₃, OCH₃O(CH₂)_(n)CH₃, O(CH₂)_(n)NH₂ or O(CH₂)_(n)CH₃ where n is from 1 to about 10; C₁ to C₁ lower alkyl,substituted lower alkyl, alkaryl or aralkyl; Cl; Br; CN; CF₃; OCF₃; O,S, or N-alkyl; O, S, or N-alkenyl; SOCH₃; SO₂CH₃; ONO₂; NO₂; N₃; NH₂;heterocycloalkyl or alkaryl; aminoalkylamino; polyalkylamino;substituted silyl: an RNA cleaving group; a cholesteryl group; aconjugate; a reporter group; an intercalator; a group for improving thepharmacokinetic properties of an oligonucleotide; or a group forimproving the pharmacodynamic properties of an oligonucleotide and othersubstituents having similar properties. Oligonucleotides having sugarmimetics such as cyclobutyls in place of the pentofuranosyl group areuseful in the present invention. Other preferred embodiments may includeat least one modified base form or “universal base” such as inosine.

Synthesis of Antisense Olionucleotides

[0076] The oligonucleotides used in accordance with this invention maybe conveniently and routinely made through the well-known technique ofsolid phase synthesis on automated nucleic acid synthesizers, such asthe Applied Biosystems 380B DNA Synthesizer which utilizes β-cyanoethylphosphoramidite chemistry. Alternatively, the oligonucleotides of theinvention may be synthesized by any of the known chemicaloligonucleotide synthesis methods. Such methods are generally described,for example in Winnacker, From Genes to Clones: Introduction to GeneTechnology. VCH Verlagsgesellshaft mbH (H. Ibelgaufts trans. 1987).

[0077] Any other means for such synthesis may also be employed; theactual synthesis of the oligonucleotides is well within the talents ofthe routineer. It is also well known to use similar techniques toprepare other oligonucleotides such as phosphorothioates and alkylatedderivatives. For example, Formula I oligonucleotides in which one ormore X is S are prepared by published procedures which are incorporatedherein by reference. Stec., W. J. et al J. Am. Chem. Soc. (1984)106:6077-6079; Adams, S. P. et al. J. Am. Chem. Soc. (1983) 105:661;Caruthers, M. H., et al, Genetic Engineering, Settlow, J. Hollander. A.Eds; Plenum Press: New York (1982) 4:1 Broido, M. S. et al; BiochemRiophys. Res. Commun. (1984) 119:663. It is also well known to usesimilar techniques and commercially available modified amidite andcontrolled pore glass (CPG) products such as biotin, fluorescein,acridine and psoralen-modified amidites and/or CPG to synthesizefluorescently labeled, biotinylated or other modified oligonucleotides.

[0078] Since the complete gene sequence of certain mamalian angiogeninsare known, including human and mouse, oligonucleotides hybridizable withany portion of the gene sequence or the mRNA transcript may be preparedby the oligonucleotide synthesis methods known to those skilled in theart.

Dosage and Administration

[0079] Overall, it is preferred to administer oligonucleotides oranalogs thereof to mammals suffering from the effects of abnormalangiogenesis, such as tumor growth, in either native form or suspendedin a carrier medium in amounts and upon treatment schedules which areeffective to therapeutically treat the mammals to reduce the effects ofabnormal angiogenesis. One or more different oligonucleotides or analogsthereof targeting different sections of the nucleic acid sequence ofangiogenin may be administered together in a single dose or in differentdoses and at different amounts and times depending upon the desiredtherapy. The oligonucleotides can be administered to mammals in a mannercapable of getting the oligonucleotides initially into the blood streamand subsequently into cells, or alternatively in a manner so as todirectly introduce the oligonucleotides into the cells or groups ofcells, for example tumor cells, by such means by electroporation or bydirect injection into the tumor. Oligonucleotides whose presence incells can inhibit transcription or protein synthesis can be administeredby intravenous injection, intravenous drip, subcutaneous,intraperitoneal or intramuscular injection, orally or rectally. Humanpharmacokinetics of certain antisense oligonucleotides have beenstudied. See Zhang et al. Clinical Pharmacology & Therapeutics (1995)58(1), 44-53 incorporated by reference in its entirety. It is within thescale of a person's skill in the art to determine optimum dosages andtreatment schedules for such treatment regimens.

[0080] Doses of the oligonucleotides or analogs thereof of the presentinvention in a pharmaceutical dosage unit will be an efficacious,nontoxic quantity selected from the range of 0.1-100 mg/kg of bodyweight, preferably 0.1-50 mg/kg and more preferably 0.1 to 25 mg/kg. Theselected dose is administered to a human patient in need of inhibitionof angiogenin expression from 1-6 or more times daily or every otherday. Dosage is dependent on severity and responsiveness of the effectsof abnormal angiogenesis to be treated, with course of treatment lastingfrom several days to months or until a cure is effected or a reductionof the effects is achieved. Oral dosage units for human administrationgenerally use lower doses. The actual dosage administered may take intoaccount the size and weight of the patient, whether the nature of thetreatment is prophylactic or therapeutic in nature, the age, weight,health and sex of the patient, the route of administration, and otherfactors.

[0081] Pharmaceutical compositions may contain suitable excipients andauxiliaries which facilitate processing of the oligonucleotides intopreparations which can be used pharmaceutically. Preferably, thepreparations, particularly those which can be administered orally andwhich can be used for the preferred type of administration, such astablets, dragees, and capsules, and preparations which can beadministered rectally, such as suppositories, as well as suitablesolutions for administration parenterally or orally, and compositionswhich can be administered bucally or sublingually, including inclusioncompounds, contain from about 0.1 to about 99 percent by weight ofactive ingredients, together with the excipient.

[0082] The pharmaceutical preparations of the present invention aremanufactured in a manner which is itself well known in the art. Forexample, the pharmaceutical preparations may be made by means ofconventional mixing, granulating, dragee-making, dissolving, orlyophilizing processes. The process to be used will depend ultimately onthe physical properties of the active ingredient used.

[0083] Suitable excipients are, in particular, fillers such as sugars,for example, lactose or sucrose, mannitol or sorbitol, cellulosepreparations and/or calcium phosphates, for example, tricalciumphosphate or calcium hydrogen phosphate as well as binders such asstarch, paste, using, for example, maize starch, wheat starch, ricestarch, potato starch, gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethylcellulose, sodium carboxymethylcellulose, and/orpolyvinyl pyrrolidone. If desired, disintegrating agents may be added,such as the above-mentioned starches as well as carboxymethyl-starch,cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a saltthereof, such as sodium alginate. Auxiliaries are flow-regulating agentsand lubricants, for example, such as silica, talc, stearic acid or saltsthereof, such as magnesium stearate or calcium stearate, and/orpolyethylene glycol. Dragee cores may be provided with suitable coatingswhich, if desired, may be resistant to gastric juices. For this purpose,concentrated sugar solutions may be used, which may optionally containgum arabic, talc, polyvinylpyrrolidone, polyethylene, glycol, and/ortitanium dioxide, lacquer solutions, and suitable organic solvents orsolvent mixtures. In order to produce coatings resistant to gastricjuices, solutions of suitable cellulose preparations such asacetyl-cellulose phthalate or hydroxypropylmethycellulose phthalate, areused. Dyestuffs and pigments may be added to the tablets of drageecoatings, for example, for identification or in order to characterizedifferent combinations of active compound doses.

[0084] Other pharmaceutical preparations which can be used orallyinclude push-fit capsules made of gelatin, as well as soft, sealedcapsules made of gelatin and a plasticizer such as glycerol or sorbitol.The push-fit capsules can contain the active compounds in the form ofgranules which may be mixed with filters such as lactose, binders suchas starches, and/or lubricants such as talc or magnesium stearate andoptionally, stabilizers. In soft capsules, the active compounds arepreferably dissolved or suspended in suitable liquids, such as fattyoils, liquid paraffin, or liquid polyethylene glycols. In additions,stabilizers may be added.

[0085] Possible pharmaceutical preparations which can be used rectallyinclude, for example, suppositories, which consist of a combination ofthe active compounds with a suppository base. Suitable suppository basesare, for example, natural or synthetic triglycerides, paraffinhydrocarbons, polyethylene glycols or higher alkanols. In addition, itis also possible to use gelatin rectal capsules which consist of acombination of the active compounds with a base. Possible base materialsinclude, for example liquid triglycerides, polyethylene glycols, orparaffin hydrocarbons.

[0086] Suitable formulations for parenteral administration includeaqueous solutions of the active compounds in water-soluble orwater-dispersible form. In addition, suspensions of the active compoundsas appropriate oily injection suspensions may be administered. Suitablelipophilic solvents or vehicles include fatty oils, for example, ethyloleate or triglycerides. Aqueous injection suspensions may containsubstances which increase the viscosity of the suspension including, forexample, sodium carboxymethyl cellulose, sorbitol, and/or dextran.Optionally, the suspension may also contain stabilizers.

[0087] Additionally, oligonucleotides of the present invention may alsobe administered encapsulated in liposomes or immunoliposomes, which arepharmaceutical compositions wherein the active ingredient is containedeither dispersed or variously present in corpuscles consisting ofaqueous concentric layers adherent to lipidic layers. Liposomes areespecially active in targeting the oligonucleotides to liver cells. Theactive ingredient, depending upon its solubility, may be present both inthe aqueous layer and in the lipidic layer, or in what is generallytermed a liposomic suspension. The hydrophobic layer, generally but notexclusively, comprises phospholipids such as lecithin and sphingomyelin,steroids such as cholesterol, more or less ionic surfactants such asdicetylphosphate, stearylamine, or phosphatidic acid, and/or othermaterials of a hydrophobic nature. The diameters of the liposomesgenerally range from about 15 nm to about 5 microns.

Antisense in Combination with Other Therapies

[0088] Published pharmacologic data indicate that phosphorothioatederivatives of oligonucleotides, when administered systemically, aretaken up preferentially by the liver (and additionally by the kidney andbone marrow). Angiogenin is known to be a normal component of humanplasma and serum synthesized predominantly by the adult liver.Therefore, oligonucleotides effective to inhibit the expression ofangiogenin should accumulate in the liver and inhibit the endogenoussynthesis of angiogenin and consequently lower its concentration inplasma and serum. The lower plasma/serum levels of angiogenin shouldthen allow for more effective antitumor therapy using any of theangiogenin binding agents described herein that inhibit angiogenin'sfunction by directly binding to the protein, since they will not have tofirst overcome binding to endogenous, circulating angiogenin beforereaching the tumor itself The use of oligonucleotides to inhibit theexpression of angiogenin in combination with other angiogenin bindingagents also lowers the potential for toxicity that might result fromsubstantial amounts of circulating angiogenin inhibitor complexes due tothe reduced amount of circulating angiogenin.

[0089] The oligonucleotides of the present invention are also envisionedto be useful in combination with other tumor targeted therapeuticmaneuvers such as chemotherapy, immunotherapy, radiation therapy and thelike so as to increase the overall anticancer therapeutic efficacy.

Oligonucleotides as Diagnostic Agents

[0090] The oligonucleotides of the present invention are also useful fordetection and diagnosis of angiogenin in clinical samples. For example,radio labeled oligonucleotides can be prepared by ³²P labeling at the 5′end with polynucleotide kinase. Sambrook et al. Molecular Cloning. ALaboratory Manual, Cold Spring Harbor Laboratory Press, 1989, Volume 2,pg. 10.59. Radio labeled oligonucleotides are then contacted with tissueor cell samples suspected of containing target nucleic acids and thesample is washed to remove unbound oligonucleotide. Radioactivityremaining in the sample indicates bound oligonucleotide (which in turnindicates the presence of target nucleic acids) and can be quantitatedusing a scintillation counter or other routine means. Abnormally highlevels of target nucleic acids can be detected in this way. Radiolabeled oligonucleotides can also be used to perform autoradiography oftissues to determine the localization, distribution and quantitation oftarget nucleic acids for research, diagnostic or therapeutic purposes.In such studies, tissue sections are treated with radio labeledoligonucleotide and washed as described above, then exposed tophotographic emulsion according to routine autoradiology procedures. Theemulsion, when developed, yields an image of silver grains over theregions expressing angiogenin.

[0091] Analogous assays for fluorescent detection of angiogeninexpression can be developed using oligonucleotides of the inventionwhich are conjugated with fluorescein or other fluorescent tag insteadof radio labeling. Such conjugations are routinely accomplished duringsolid phase synthesis using fluorescently labeled amidites or CPG (c.g.fluorescein labeled amidites and CPG available from Glen Research,Sterling Va. Sec. 1993 Catalog of Products for DNA Research, GlenResearch, Sterling Va, p. 21).

[0092] Each of these assay formats is known in the art. One of skillcould easily adapt these known assays for detection of target nucleicacids in accordance with the teachings of the invention providing anovel and useful means to detect levels of nucleic acids encodingangiogenin.

[0093] The following examples are set forth as representative of thepresent invention. These examples are not to be construed as limitingthe scope of the invention as these and other equivalent embodimentswill be apparent in view of the present disclosure, figures, tables, andaccompanying claims.

EXAMPLE 1 Materials

[0094] Materials used in the following experimental examples wereobtained as follows. Human PC-3 prostate and HT-29 colon tumor cellswere obtained from the American Type Culture Collection. MDA-MB435 humanbreast tumor and PC-3M human prostate tumor cell lines were obtainedfrom Dr. Isaiah J. Fidler (M.D. Anderson Cancer Center). Human MCF-7breast cancer cells were obtained from Dr. Marc Lippman (GeorgetownUniversity Medical Center). Cell culture supplies were obtained asfollow: all tissue culture plastics were from Costar; Dulbecco'smodified Eagle's medium (DMEM), Ham's F-12 medium, MEM Eagle medium,trypsin-versene, and Hanks' buffered salt solution (HBSS) were obtainedfrom BioWhittaker; fetal bovine serum (FBS) was from Hyclone. Materialsfor the enzyme-linked immunosorbent assay (ELISA) were as follows: humanangiogenin was isolated from an Escherichia coli expression system[Shapiro, R, Harper, J. W., Fox, E. A., Jansen, H -W., Hein, F., andUhlmann, E. (1988) Anal. Biochem. 175, 450-461] and was provided by Dr.Robert Shapiro (Harvard Medical School); the anti-human angiogenin mAb26-2F was obtained by us as described (Mahadevan and Hart, 1990, supra);the rabbit polyclonal anti-human angiogenin antibody R113 was producedby immunization into a rabbit of human angiogenin together with Freund'sadjuvant using classical techniques; ELISA plates were from Costar;bovine serum albumin (BSA) and p-nitrophenyl phosphate were from Sigma;alkaline phosphatase-labeled goat anti-rabbit IgG was obtained fromKirkegaard and Perry. Lipofectin was from GibcoBRL. Slow release pelletscontaining 17 β-estradiol were obtained from Innovative Research ofAmerica. Custom-synthesized angiogenin sense and antisense [S]ODNs werefrom Promega or Boston BioSystems. Outbred, male and female athymic(nu/nu) mice were obtained from Charles River Laboratories andmaintained under specific pathogen-free conditions in an environmentstrictly controlled for temperature and humidity. Matrigel basementmembrane matrix was from Collaborative Biomedical Products.

EXAMPLE II Cell Culture Growth Conditions

[0095] Cell cultures used in the following experimental examples aredescribed as follows. All cells were maintained at 37° C. in ahumidified, 95% air/5% CO₂ environment. IIT-29 cells were grown in DMEMcontaining 5% FBS; PC-3M cells were grown in MEM Eagle medium containing10% FBS and vitamins; PC-3 cells were cultured in Ham's F-12 containing7% FBS. All growth medium was supplemented with 2 mM L-glutamine andantibiotics (gentamicin and fungizone). For experiments, cells wereharvested with trypsin-versene and counted with either a Coulter counteror by hemacytometry following staining with Trypan blue for viabilitydetermination. Prior to injection into mice, cells were first washedtwice with HBSS.

EXAMPLE III Measurement of Angiogenin Levels

[0096] Angiogenin levels in medium conditioned by human tumor cells weremeasured by a double antibody ELISA as described [Newton, D. L., Xue,Y., Olson, K. A, Fett, J. W., and Rybak, S. M. (1996) Biochemistry 35,545-553]. Antihuman angiogenin nAb 26-2F was coated onto wells of anELISA plate and blocked with BSA. Dilutions of medium to be tested werethen added to the plate and incubated overnight. After washing, rabbitanti-human angiogenin antibody (R113) was added. Bound R113 was detectedby adding alkaline phosphatase-labeled goat anti-rabbit IgG followed bythe addition of p-nitrophenyl phosphate. The plates were read at 405 nmwith a computer-controlled Bio-Tek EL 311 ELISA reader using theassociated data analysis program. Angiogenin levels in conditionedmedium were quantitated by comparison with a standard curve of humanangiogenin.

EXAMPLE IV Antisense Oligodeoxynucleotides

[0097] Angiogenin sense and antisense phosphorothioateoligodeoxynucleotides, [S]ODNs, used in the following experiments wereas follows. Two antisense 18-mer [S]ODNs were custom-synthesized byPromega based on the nucleic acid sequences of the angiogenin geneencompassing the AUG initiation codon and transcriptional start siteregions and labeled JF2S and JF4S, respectively. In addition, an 18-mercontrol sense [S]ODN complementary to JF2S was synthesized and labeledJF1S. Their compositions are: JF1S 5′-GAAGAGATGGTGATGGGC-3′ JF2S5′-GCCCATCACCATCTCTTC-3′ JF4S 5′-ACACGOCATCATGAATCA-3′

[0098] Other preferred oligonucleotides include the following: JF6S5′-CCAGGGGCCCGCTGGTA-3′ JF8S 5′-ACCAAATTTTATATTCTA-3′ JFlOS5′-CAGGCCCATCACCATCAC-3′ JF12S 5′-GCCCAGOCCCATCACCAT-3′ JF13S5′-TCTCTGACACGGCATCAT-3′

[0099] JF6S encompasses the 3′-termination site, JF8S encompasses the5′-“TATA” box site, JF10S and JF12S encompass the “AUG” translationalstart site and comprise variations of sequence from JF2S, and JF13Sencompasses the 5′-transcriptional start site and comprises a variationof sequence from JF4S.

[0100] It is to be understood that additional oligonucleotides withinthe scope of the present invention can be prepared by first selecting atarget sequence anywhere along the known nucleic acid sequence of theangiogenin gene. An oligonucleotide complementary to the target sequencecan then be prepared based upon the known complementary relationshipbetween nucleic acids. In this manner, any number of oligonucleotidescomplementary to target sequences of the angiogenin gene can be preparedand their ability to inhibit the expression of the angiogenin gene canthan be determined based upon the teachings presented herein.

EXAMPLE V Antisense Oligodeoxynucleotides Inhibit Expression ofAngiogenin

[0101] Experiments initially performed in vitro were aimed at assessingwhether these angiogenin antisense reagents were effective inhibitors ofangiogenin synthesis by prostatic carcinoma cell lines. Efficienttransfection of ODNs in vitro requires the presence of a cationic lipid,one of which, lipofectin, was obtained from GibcoBRL. Details of thelipofectin transfection procedure are provided by the manufacturer,GibcoBRL. In a first experiment, the results of which are shown in FIG.2, panel A, PC-3 prostatic carcinoma cells (5×10⁵ cells in 35 mm dishes)were treated in vitro for 20 hr with lipofectin (5 μl) alone (control,white bar) or lipofectin plus JF2S (0. 5 μM) (black bar). The growthmedium was then replaced and the cells allowed to recover for 24 hr.After that period the cells were harvested and counted and theconditioned medium was assayed for angiogenin levels by ELISA. Theamount of angiogenin per cell number for the antisense-treated culturesin percent compared with control-treated cells (100%) is plotted. Theresults of a second in vitro experiment under the same conditions isplotted in panel B of FIG. 2. The data indicate that angiogeninproduction in vitro as a function of cell number decreased by 18-33% bytreating with the combination of lipofectin plus JF2S in comparison withthe treatment with lipofectin alone.

[0102]FIG. 3 shows the results of a similar experiment using HT-29 colonadenocarcinoma cell line. In the first experiment, the results of whichare plotted in FIG. 3 panel A, HT-29 cells (5×10⁵ cells in 35 mm dishes)were treated in vitro for 20 hr with lipofectin (5 μl) alone (control,white bar) or lipofectin plus JF2S (0.5 μM) (black bar). The growthmedium was then replaced and the cells allowed to recover for 24 hr.After that period the cells were harvested and counted and theconditioned medium was assayed for angiogenin levels by ELISA. Theamount of angiogenin per cell number for the antisense-treated culturesin percent compared with control-treated cells (100%) is plotted. Theresults of a second in vitro experiment under the same conditions isplotted in panel B of FIG. 3. The data indicates that angiogeninproduction in vitro as a function of cell number decreased by 30-38% bytreating with the combination of lipofectin plus JF2S in comparison withthe treatment with lipofectin alone.

[0103] The data demonstrates that for both PC-3 and HT-29 tumor celltypes angiogenin production in vitro as a function of cell number wasdecreased by treating with the combination of lipofectin plus JF2S incomparison to treatment with lipofectin alone.

EXAMPLE VI Antisense Oligodeoxynucleotides Reduce Tumor Size

[0104] The ex vivo-treated PC-3 tumor cells of Example V were injecteds.c. into athymic mice (2.5×10⁵ cells/mouse; 5 mice/group) with theusual co-administration of a 1:2 proportion of Matrigel for reproduciblecell growth of this cell line. After 8 days, by which time the controltumors had attained a size in excess of that supportable by diffusionand were therefore dependent upon angiogenesis, the mice were sacrificedand the tumors were excised and weighed. The average weight of thetumors resulting from injection of the antisense-treated cells inpercent was compared with that of the control group's tumors (100%) andshown in FIG. 2 (panels A & B, in vivo). Tumors arising from injectionof JF2S-treated PC-3 cells were both 31-54% smaller in average weightthan the tumors which developed from their respective control-treatedcells. Of additional importance, in the experiment represented by FIG. 2panel B, 1 mouse out of 5 did not develop an observable tumor by thetime of sacrifice.

[0105] The ex vivo-treated HT-29 tumor cells were also injected s.c.into athymic mice (2.5×10⁵ cells/mouse; 5 mice/group). After 15 days, bywhich time the control tumors had attained a size in excess of thatsupportable by diffusion and were therefore dependent upon angiogenesis,the mice were sacrificed and the tumors were excised and weighed. Theaverage weight of the tumors resulting from injection of theantisense-treated cells in percent was compared with that of the controlgroup's tumors (100%) and shown in FIG. 3 (panels A & B, in vivo).Tumors arising from injection of JF2S-treated PC-3 cells were both53-66% smaller in average weight than the tumors which developed fromtheir respective control-treated cells. Of additional importance, in theexperiment represented by FIG. 3 panel A, 1 mouse out of 5 did notdevelop an observable tumor by the time of sacrifice.

[0106] These results indicate that a correlation exists betweendecreased tumor growth in vivo and decreased angiogenin production bytumor cells treated in vitro with JF2S.

[0107]FIGS. 4, 5, 6 and 7 show the results of in vitro experiments inwhich HT-29, PC-3, MDA-MB435 or PC-3M tumor cells, respectively, weretreated with lipofectin alone or lipofectin with either antisense [S]ODNJF2S or control sense [S]ODN JF1 S. The amount of angiogenin per cellnumber for the antisense-treated (black bar) and sense-treated (greybar) cultures in percent compared with control lipofectin-treated cells(100%) (white bar) is plotted. Angiogenin production in vitro as afunction of cell number decreased by 39% (HT-29), 65% (PC-3), 45%(MDA-MB-435) and 48% (PC-3M) by treating with the combination oflipofectin plus antisense [S]ODN JF2S in comparison with treatment withlipofectin alone. Treatment with control sense [S]ODN JIF1S pluslipofectin resulted in a decrease of 13% (HT-29), 44% (PC-3), 17%(MDA-MB-435) and 26% (PC-3M) in comparison with treatment withlipofectin alone. These same ex vivotreated tumor cells weresubsequently injected into athymic mice [HT-29 cells: 2.5×10⁵cells/mouse (5 mice/group); PC-3 cells: 1.25×10⁵ cells/mouse injecteds.c., mixed with a 1:2 proportion of Matrigel (5 mice/group); MDA-MB-435cells: 7.5×10⁵ cells/mouse, injected s.c. into the mammary fat pad (5mice/group, except for the group receiving the antisense [S]ODNJF2S-treated cells, in which there were 7 mice); PC-3M: 2.5×10⁵cells/mouse injected s.c., mixed with a 1:2 proportion of Matrigel (5mice/group)]. After 17 (HT-29), 25 (PC-3), 30 (MDA-MB-435) or 17 (PC-3M)days, by which time the control tumors had attained a size in excess ofthat supportable by diffusion and were therefore dependent uponangiogenesis, the mice were sacrificed and the tumors were excised andweighed. The average weight of the tumors resulting from injection ofantisense [S]ODN JF2S-treated cells in percent was compared with that ofthe control group's tumors (100%) and shown in FIG. 4 (HT-29), FIG. 5(PC-3), FIG. 6 (MDA-MB-435) and FIG. 7 (PC-3M) (in vivo). Tumors arisingfrom injection of antisense [S]ODN JF2S-treated tumor cells were 53%(HT-29), 92% (PC-3), 59% (MDA-MB-435) and 74% (PC-3M) smaller in averageweight than the tumors which developed from the cells treated withlipofectin alone. Tumors arising from PC-3 cells treated with thecontrol sense [S]ODN JF1S were 16% (FIG. 5, in vivo) smaller than thosetumors which developed from cells treated with lipofectin alone. Tumorsarising from IHT-29, MDA-MB435 or PC-3M cells treated with the controlsense [S]ODN JF1S were actually 14%, 4% or 21% larger, respectively,than those tumors which developed from the cells treated with lipofectinalone. In all three experiments all mice receiving either controllipofectin or sense [S]ODN JF1S-treated cells developed tumors. Amongthose mice receiving cells treated with antisense [S]ODN JF2S, tumorsdid not develop by the time of termination of the experiments in 1 outof 5 (HT-29 cells), 4 out of 5 (PC-3 cells), 1 out of 7 (MDA-MB435cells) and 2 out of 5 (PC-3M cells) mice. These results further indicatethat a correlation exists between decreased tumor growth in vivo anddecreased angiogenin production by tumor cells treated in vitro withantisense [S]ODN JF2S.

[0108]FIG. 8 is a photograph of the actual tumors excised from miceinjected with PC-3 cells treated in vitro with either the antisense[S]ODN, JF2S, (bottom row) or control lipofectin (top row) as describedin Example V and as shown in FIG. 2 panel B. This shows the differencesbetween these two groups of tumors in both size and occurrence. Onemouse in the antisense-treated group (bottom row) did not develop atumor while those that did develop were on average much smaller sizethan tumors arising from control-treated cells (top row).

[0109]FIG. 9 is a photograph of the actual tumors excised from miceinjected with HT-29 cells treated in vitro with either the antisense[S]ODN, JF2S (bottom row) or control lipofectin (top row) as describedin Example V and as shown in FIG. 3 panel B. Once again the differencesbetween these two groups of tumors in size is evident, with the tumorsdeveloping from the lipofectin plus JF2S-treated cells being muchsmaller on average than those tumors developing from HT-29 tumor cellstreated with lipofectin alone. In particular, three of the tumorsproduced by HT-29 tumor cells treated with JF2S were extremely small insize.

[0110] Photographs of the actual tumors excised from the mice inexperiments shown in FIGS. 4, 5, 6 and 7 are shown in FIGS. 10, 11, 12and 13, respectively. In each case the photograph shows the tumorsresulting from injection of cells treated in vitro with either antisense[S]ODN JF2S (bottom row), sense control [S]ODN JF1S (middle row) orcontrol lipofectin (top row). The average size of the two groups ofcontrol tumors arising from tumor cells treated with either the sense[S]JODN JF1S or lipofectin alone is essentially equivalent, while theaverage size of those tumors arising from the tumor cells treated withthe antisense [S]ODN JF2S were significantly smaller than either ofthese two control groups. In fact, tumors did not develop by thetermination of the experiments in 1 out of 5 (HT-29 cells, FIG. 10), 4out of 5 (PC-3 cells, FIG. 11), 1 out of 7 (MDA-MB435 cells, FIG. 12)and 2 out of 5 (PC-3M cells, FIG. 13) mice receiving cells treated withantisense [S]ODN JF2S.

[0111] From these studies the conclusion can again be drawn thatangiogenin is indeed critical for the growth/establishment of tumors inthis mouse model, further validating the proposition thatanti-angiogenin therapies are effective for treatment of cancerclinically.

EXAMPLE VII

[0112] In two further experiments, shown in FIG. 14 panels A and B, theamount of angiogenin produced by PC-3 cells in vitro could be furtherdecreased by slightly adjusting the conditions of transfection withantisense JF2S. The figure also shows that JF2S can additionally inhibitangiogenin production by PC-3M tumors cells and that another angiogeninantisense [S]ODN, JF4S, also can inhibit the synthesis of angiogenin byboth PC-3 and PC-3M cells in vitro. PC-3 (panel A) or PC-3M (panel B)cells were treated for 20 hr with HBSS as diluent control (white bars),lipofectin (5 μl) alone (control, single cross-hatched bars), lipofectinplus JF2S [(0.5- (black bars) or 1.0 μM(dotted bars)], or lipofectinplus JF4S [(0.5- (grey bars) or 1.0 μM (double crossed hatched bars)].The growth medium was then replaced and the cells allowed to recover for48 hr at which time the cells were harvested and counted and theconditioned medium was assayed for angiogenin levels by ELISA. Theamount of angiogenin per cell number for each group in percent comparedto that of the HBSS-treated control group (100%) is plotted. This showsthat treatment with antisense JF2S, under these conditions, was now ableto inhibit the synthesis of angiogenin by PC-3 cells by about 87% ascompared with HBBBS-treated cells (panel A). JF2S also substantiallyinhibits angiogenin production by a third tumor cell line PC-3M (panelB). Panels A & B also show that a second angiogenin antisense reagentdirected toward the transcriptional start site of the angiogenin gene,JF4S, also effectively interferes with angiogenin production by bothPC-3 and PC-3M tumor cells. Lipofectin alone has essentially no effecton angiogenin levels secreted by either of the two cell types (panels A& B). Importantly, treatment with a control “sense” sequence [S]ODNcomplementary to JF2S, i.e. JF1S, did not result in decreased angiogeninproduction by PC-3 cells (not shown).

EXAMPLE VIII

[0113]FIG. 15 shows the results obtained in three separate therapyexperiments. Each experiment was conducted as follows. On day 0,mixtures of PC-3 tumor cells (1×10⁴ cells/mouse) with either antisense[S]ODN JF2S (200 μg/mouse), control sense [S]ODN JF1S (200 μg/mouse) orPBS (as diluent control) were injected s.c., together with a 1:2proportion of Matrigel to other components into male athymic mice.Treatment was continued for 48 days as follows: day 1-6, antisense[S]ODN JF2S (100 μg/mouse), control sense [S]ODN JF1 S (100 μg/mouse) orPBS as diluent control injected daily s.c. 6 times per week; days 7-20:antisense [S]ODN JF2S (50 μg/mouse), control sense [S]ODN JF1S (50μg/mouse) or PBS as diluent control injected daily s.c. 6 times perweek; days 21-49: antisense [S]ODN JF2S (50 μg/mouse), control sense[S]ODN JF IS (50 μg/mouse) or PBS as diluent control injected daily s.c.4 times per week. Mice were examined twice a week for the presence of apalpable tumor. After day 49, treatment was stopped.

[0114]FIG. 15, panels A, B, and C show the percentage of mice bearing apalpable tumor in each of the antisense [S]ODN JF2S-treated (black bar)and control sense [S]ODN JFIS-treated (grey bar) groups compared withthe PBS diluent control-treated group (white bar, 100%) for threeseparate, independent experiments. In experiment 1 (panel A), no tumorswere observed in any of the antisense [S]ODN JF2S-treated mice as of day65, the day at which the mice were sacrificed. In contrast, all of themice treated with either control sense [S]ODN JF1 S or PBS as diluentcontrol exhibited palpable tumors at the time of sacrifice on day 65. Inexperiment 2, the mice treated with control sense [S]ODN JF1S or PBS allexhibited tumors by day 49, and were subsequently sacrificed. However,no tumors were observed at that time in any of the mice treated with theantisense [S]ODN JF2S. These mice were kept for observation with nofurther treatment until day 276; no tumors were observed in any of thesemice during this period. In experiment 3, the mice treated with controlsense [S]ODN JF1 S or PBS also all exhibited tumors by day 49 and werethen sacrificed. The mice treated in this experiment with antisense[S]JODN JF2S did not develop tumors during a subsequent observationperiod, without further treatment, until sacrifice on day 139. Inexperiments 1 and 2 the [S]ODNs used for treatment were prepared byPromega Corp., while in experiment 3 the [S]ODNs were prepared by BostonBioSystems. Thus in three separate experiments treatment with theantisense [S]ODN JF2S was shown to prevent the appearance of PC-3 humantumors after injection of these tumor cells into athymic mice.

[0115]FIGS. 16 and 17 show the results of similar in vivo therapyexperiments using human breast tumor MDA-MB435 or MCF-7 cells,respectively. The former cell line is estrogen-independent, while thelatter is estrogen-dependent. In these experiments the source of the[S]ODNs used for therapy was Boston BioSystems. Tumor cells (5×10⁵MDA-M435 cells/mouse or 2×10⁶ MCF-7 cells/mouse) were injected into thesurgically-exposed mammary fat pad behind the left front leg of femaleathymic mice. In the case of the estrogen-dependent MDF-7 cell line aslow release (60 day) estrogen pellet containing 0.72 mg of 17β-estradiol was inserted s.c. within 1 cm of the area of tumor cellinjection as an exogenous source of estrogen. Within 5 minutes of thetumor cell injection, mice were treated by s.c. injection in the area ofthe tumor cell injection with either antisense [S]ODN JF2S (200μg/mouse), control sense [S]ODN JF1S (200 μg/mouse) or PBS as diluentcontrol. The mice were subsequently treated daily 6 times per week witheither PBS as diluent control or antisense [S]ODN JF2S or control sense[S]ODN JF1S (100 μg of each [S]ODN/mouse in the experiment usingMDA-MB435 cells and 200 μg of each [S]ODN/mouse in the experiment usingMCF-7 cells). The mice were checked for palpable tumors twice a weekuntil sacrifice on day 28. FIG. 16 shows that all PBS diluent control(open circles)-and control sense [S]ODN JF1S (closed circles)-treatedmice developed palpable tumors by day 17. In contrast, at that timetumors had developed in only 29% of the antisense [S]ODN JF2S (closedsquares)-treated mice by day 17. On day 28, the day of termination ofthe experiment, 40% of the antisense [S]ODN JF2S-treated mice were stilltumor-free. FIG. 17 shows that 100% of the PBS diluent control (opencircles)-and control sense [S]ODN JF1 S (closed circles)-treated micedeveloped palpable tumors by day 28, while only 22% of those micetreated with antisense [S]ODN JF2S (closed squares) exhibited palpabletumors at the time. Thus in vivo treatment with antisense [S]ODN JF2Sdelayed and in a subset of mice completely prevented the appearance oftumors from two different human breast tumor cell lines injected intoathymic mice.

EXAMPLE IX

[0116] The efficacy of antisense [S]ODN JF2S in preventing tumormetastasis was investigated using an orthotopic model of human prostatecancer metastasis in athymic mice. Orthotopic tumor models are those inwhich tumor cells are implanted into the mouse organ equivalent to thesource organ from which the tumor cell line is derived. In the modelused to test antisense [S]JODN JF2S, the human prostate tumor cell linePC-3M (3.75×10⁵ cells/mouse) was injected into one of the surgicallyexposed lobes of the prostate gland of an athymic mouse. The mouse wasthen treated 1 hour later by i.p. injection with either antisense [S]ODNJF2S (500 μg/mouse in the high dose group or 200 μg/mouse in the lowdose group), control sense [S]ODN JF1S (500 μg/mouse in the high dosegroup or 200 μg/mouse in the low dose group), anti-angiogenin monoclonalantibody 26-2F (300 μg/mouse; included as a positive control treatmentgroup, since it has been previously determined that this monoclonalantibody is efficacious in preventing PC-3M tumor metastasis in the samemodel), or PBS as diluent control. The [S]ODN-and PBS-treated mice weresubsequently injected i.p. with the same materials at the same abovedoses per mouse daily 6 times per week from day 1-13, followed byinjections of the same dose of the same materials 4 times per week untilday 38. Monoclonal antibody 26-2F was administered on the same schedulebut at a previously determined optimal dose of 180 μg/mouse for days1-38. On day 39 the mice were sacrificed and the prostate examined forevidence of tumor. At that time all mice in the experiment contained aprimary tumor in their prostate gland. The regional iliac lymph nodeswere removed and preserved in phosphate-buffered formalin. Thesepreserved lymph nodes were later dehydrated, embedded in paraffin, cutinto 4 mm sections and stained with hematoxylin and eosin. The slideswere then examined by a pathologist in a blinded fashion for evidence ofmetastasis. Table 2 below shows the results of this examination in termsof the number of mice in the indicated treatment group harboringmetastasis in at least one of the two iliac lymph nodes divided by thetotal number of mice in the treatment group. This number is expressed asa percentage in parentheses below the aforementioned fraction. TABLE 2Incidence of Metastasis mAb Sense Sense PBS 26-2F control Antisensecontrol Antisense (diluent (medium JF1S JF2S JF1S JF2S control) dose)(high dose) (high dose) (low dose) (low dose) {fraction (6/6)} {fraction(4/9)} {fraction (9/9)} {fraction (5/10)} {fraction (6/6)} ⅘ (100%)(44%) (100%) (50%) (100%) (80%)

[0117] All of the mice treated with PBS, a diluent control, or controlsense [S])ODN JF1S (at both high and low doses) developed metastasis inat least one of the regional iliac lymph nodes. Monoclonal antibody26-2F protected 56% of the mice from developing metastasis in theregional lymph nodes, a percentage comparable to that obtained inprevious experiments. A low dose of antisense [S]ODN JF2S protected 1out of the 5 mice from developing metastasis. However, the high dose ofantisense [S]ODN JF2S protected 50% of the mice from forming regionallymph node metastasis. Thus antisense [S]ODN JF2S is effective inpreventing human tumor metastasis in an orthotopic model of prostatetumor metastasis.

[0118] It is to be understood that the embodiments of the presentinvention which have been described are merely illustrative of some ofthe applications of the principles of the invention. Numerousmodifications may be made by those skilled in the art based upon theteachings presented herein without departing from the true spirit andscope of the invention.

What is claimed is:
 1. A compound for inhibiting expression ofangiogenin comprising an oligonucleotide or analog thereof having a basesequence complementary to a target portion of a nucleic acid encodingangiogenin.
 2. The compound of claim 1 wherein the base sequence isconfigured to bind to the target portion of the nucleic acid in a mannerto inhibit the expression of angiogenin.
 3. The compound of claim 2wherein the oligonucleotide analog comprises a modified internucleotidelinkage, a modified purine or pyrimidine moiety, a modified sugarmoiety, a modified 5′ hydroxyl moiety, a modified 3′ hydroxyl moiety ora modified 2′ hydroxyl moiety.
 4. The compound of claim 3 wherein themodified internucleotide linkage comprises a substituent having animproved aqueous or lipid solubility or improved resistance to nucleasedigestion.
 5. The compound of claim 4 wherein the modifiedinternucleotide linkage is selected from the group consisting ofphosphorothioate, alkyl or cycloalkyl phosphorothioate, N-alkyl orcycloalkyl phosphoramidates, phosphorodithioates, alkyl or cycloalkylphosphonates, phosphodiester, phosphotriester, C₁- C₄ alkyl, cycoaloyl,short chain heteroatomic or heterocyclic backbone, morpholino backbone,polyprotein-nucleic acid or peptide-nucleic acid backbone, polyamide,CH₂—NH—O—CH₂, CH₂—N(CH₃)—O—CH₂, CH₃—O—N(CH₃)—CH₂, CH₂—N(CH₃)—CH₂ andO—N(CH₃)—CH₂—CH₂.
 6. The compound of claim 3 wherein the modified purineor pyrimidine moiety includes inosine.
 7. The compound of claim 3wherein the modified sugar moiety includes sugar mimetics comprisingC₄-C₈ cycloalkyl.
 8. The compound of claim 3 wherein the modified 5′ or3′ hydroxyl moiety is selected from the group consisting of C₁₋₄ alkoxy,intercalating agent, peptide, enzyme, ribozyme, substituted acridine,2-methoxy-6-chloro-9-pentylaminoacridine,N-(6-chloro-2-methoxyacridinyl)-0-methoxydisopropylaminophosphinyl-3-aminopropanoland N-(6chloro-2-methoxyacridinyl)-O-methoxydisopropylaminophosphinyl-5-aminopentanol.9. The compound of claim 1 wherein the modified 2′ hydroxyl moiety isselected from the group consisting of OH, SH, SCH₂, OCH₃, F, OCN,OCH₆CH₃, OCH₃OCH₃, OCH₃O(CH₂)_(n)CH₃, O(CH₂)_(n)NH₂ or O (CH₂)_(n)CH₃where n is from 1 to about 10; C₁ to C₁₀ lower allyl, substituted loweralkyl, alkaryl or aralkyl; Cl; Br; CN; CF₃; OCF₃; O, S, or N-alkyl; O,S, or N-alkenyl; SOCH₃; SO₂CH₃; ONO₂; NO₂; N₃; NH₂; heterocycloalkyl oralkaryl; aminoalkylamino; polyalkylamino; substituted silyl: an RNAcleaving group; a cholesteryl group; a conjugate; a reporter group; anintercalator; a group for improving the pharmacokinetic properties of anoligonucleotide; and a group for improving the pharmacodynamicproperties of an oligonucleotide.
 10. The compound of claim 1 whereinthe base sequence of the oligonucleotide or analog thereof is selectedfrom the group consisting of 5′-GCCCATCACCATCTCTTC-3′,5′-ACACGGCATCATGAATCA-3′, 5′-CCAGCGGCCCGCTGGTTA-3′,5′-ACCAAATTTTATATTCTA-3′, 5′-CAGGCCCATCACCATCAC-3′,5′-GCCCAGGCCCATCACCAT-3′, and 5′-TCTCTGACACGGCATCAT-3′.


11. A composition for inhibiting expression of angiogenin comprising aneffective amount of an oligonucleotide or analog thereof having a basesequence complementary to a target portion of a nucleic acid encodingangiogenin in a pharmaceutically acceptable carrier.
 12. The compositionof claim 11 wherein the base sequence of the oligonucleotide or analogthereof is selected from the group consisting of
 13. A compound forinhibiting expression of angiogenin having the

wherein X is O, S, or C₁₋₄ alkyl; B is adenine, guanine, cytosine, orthymine selected such that the oligonucleotide has a complementary basesequence with a portion of a target nucleic acid strand coding forangiogenin thereby inhibiting expression thereof; R₁ is H, C₁₋₄ alkyl,intercalating agent, peptide, enzyme, ribozyme, substituted acridine,2-methoxy-6-chloro-9-pentylaminoacridine,N-(6-chloro-2-methoxyacridinyl)-O-methoxydisopropylaminophosphinyl-3-aminopropanoland N-(6chloro-2-methoxyacridinyl)-O-methoxydisopropylaminophosphinyl-5-aminopentanol.or substituted acridine; R₂ is H, OH, SH, SCH₂, OCH₃, F, OCN, OCH₆CH₃,OCH₃OCH₃, OCH₃O(CH₂)_(n)CH₃, O(CH₂)_(n)NH₂ or O(CH₂)_(n)CH₃ where n isfrom 1 to about 10; C₁ to C₁₀ lower alkyl, substituted lower alkyl,alkaryl or aralkyl; Cl; Br; CN; CF₃; OCF₃; O, S, or N-alkyl; O, S, orN-alkenyl; SOCH₃; SO₂CH₃; ONO₂; NO₂; N₃; NH2; heterocycloalkyl oralkaryl; aminoalkylamino; polyalkylamino; substituted silyl: an RNAcleaving group; a cholesteryl group; a conjugate; a reporter group; anintercalator; a group for improving the pharmacokinetic properties of anoligonucleotide; or a group for improving the pharmacodynamic propertiesof an oligonucleotide; and n is 5to
 100. 14. The compound of claim 13wherein the base sequence is selected from the group consisting of5′-ACACGOCATCATGAATCA-3′, 5′-CCAGGGOCCCGCTGGTTA-3′,5′-ACCAAATTTTATATTCTA-3′, 5′-CAGOCCCATCACCATCAC-3′,5′-GCCCAG6CCCATCACCAT-3′, and 5′-TCTCTGACACGGCATCAT-3′.


15. A method for inhibiting expression of angiogenin in a mammalcomprising administering to the mammal an effective amount of anoligonucleotide or analog thereof having a base sequence complementaryto a target portion of a nucleic acid encoding angiogenin so as toinhibit the expression of angiogenin.
 16. A method for reducing size oftumors associated with angiogenesis in a mammal comprising administeringto the mammal an effective amount of an oligonucleotide or analogthereof having a base sequence complementary to a target portion of anucleic acid encoding angiogenin so as to reduce tumor size.
 17. Amethod for decreasing production of angiogenin in a mammal comprisingadministering to the mammal an effective amount of an oligonucleotide oranalog thereof having a base sequence complementary to a target portionof a nucleic acid encoding angiogenin so as to decrease production ofangiogenin.
 18. A method for inhibiting metastasis of tumor cells in amammal comprising administering to the mammal an effective amount of anoligonucleotide or analog thereof having a base sequence complementaryto a target portion of a nucleic acid encoding angiogenin so as toinhibit metastasis of tumor cells.
 19. A method for inhibiting theestablishment of tumor cells in a mammal comprising administering to themammal an effective amount of an oligonucleotide or analog thereofhaving a base sequence complementary to a target portion of a nucleicacid encoding angiogenin so as to inhibit establishment of tumor cells.20. A method for inhibiting growth of tumors associated withangiogenesis in a mammal comprising administering to the mammal aneffective amount of an oligonucleotide or analog thereof having a basesequence complementary to a target portion of a nucleic acid encodingangiogenin so as to inhibit tumor growth.
 21. A method for detecting thepresence of angiogenin in a sample comprising contacting the sample witha labeled oligonucleotide or analog thereof having a base sequencecomplementary to a target portion of a nucleic acid encoding angiogenin;allowing the labeled oligonucleotide or analog thereof to bind to thetarget portion of the nucleic acid encoding angiogenin; and detectingthe labeled oligonucleotide or analog thereof.
 22. A method fordetecting the presence of angiogenin in a mammal comprisingadministering to the mammal a labeled oligonucleotide or analog thereofhaving a base sequence complementary to a target portion of a nucleicacid encoding angiogenin; allowing the labeled oligonucleotide or analogthereof to bind to the target portion of the nucleic acid encodingangiogenin; and detecting the labeled oligonucleotide or analog thereof.23. A method for diagnosing conditions associated with abnormalangiogenesis in a mammal comprising administering to the mammal alabeled oligonucleotide or analog thereof having a base sequencecomplementary to a target portion of a nucleic acid encoding angiogenin;allowing the labeled oligonucleotide or analog thereof to bind to thetarget portion of the nucleic acid encoding angiogenin; detecting thelabeled oligonucleotide or analog thereof; measuring the labeledoligonucleotide or analog thereof; and determining the abnormalcondition based on the detecting and measuring of the labeledoligonucleotide or analog thereof.